1. Field of the Invention
The invention relates to the field of autoradiography and, more particularly, (1) to a practical, economical, and sensitive method of intensifying the effect of weak, e.g. beta, radioactive emitters, such as tritium and carbon-14, on film, (2) to a new composition of matter for carrying out the aforesaid method and (3) to the resulting autoradiogram.
2. Description of the Prior Art
When first used, the process of autoradiography depended principally on the direct action of the radiation from radioactive materials on the radiation-sensitive chemical particles embedded in the film's emulsion to provide a photo, e.g. x-ray photo, when a layer or plate containing such radioactive materials is sandwiched against the emulsion and the sandwich is exposed.
The relatively short range of penetration of weak, e.g. beta, radioactive emitters, such as tritium, requires an exceedingly and impractically long exposure time and gives poor resolution particularly when the layer containing the radioactive materials is relatively thick, as in the case of polyacrylamide electrophoresis gels which are commonly used in autoradiography.
On the other hand, this very property of weak radioactive emitters makes them safer and, hence, highly desirable for use in autoradiography.
Also, these weak emitters are the preferred radionuclides for chemical combination in organic compounds for many labelling and tracing purposes.
The detection and measurement of these low energy nuclides have been dramatically improved in the general field of radioactivity measurement with the development of highly sensitive instrumentation and with the increased use of systems utilizing scintillators or fluors, in which the radiation stimulates or excites the scintillator, resulting in light flashes or emissions sufficiently long lived to be measured and not hindered by the matter, e.g. polyacrylamide gel, through which they must travel before being detected.
This technique has been applied to autoradiography. Although called autoradiography in the broad sense, a more appropriate name is autofluorography because the radioactivity is converted to light emissions by the scintillator fluor and it is these light emissions that are photographed to give the desired measurement. It is the intensity of the light transmitted to the photographic film, which is measured to indicate the amount of radioactivity thereby indicating, e.g. the purity or amount of a radioactive component or the condition of animal tissue.
In the case of chromatography and electrophoresis, the radioactive material to be measured is absorbed or absorbed according to conventional techniques on or in an organic or inorganic absorbent or adsorbent layer or column of material, e.g. silica gel, alumina, cellulose, polyamide, polyacrylamide, cross-linked dextran, agarose, etc., which is usually supported on a plate, e.g. glass or plastic sheet. This is called a chromatogram or electrophoretogram. In the case of radioactive labelled animal tissues, e.g. tissue autoradiography, the radioactive material is usually administered to the live animal and becomes selectively absorbed or adsorbed into certain tissues so that the tissue, usually in the form of a thin slice, may be considered as the absorbent or adsorbent layer. In the case of paper chromatography the paper (cellulose) is the adsorbent.
Where the adsorbent material is in the form of a thin layer supported on a plate, it is called thin layer chromatography and a thin layer chromatogram.
Conventionally, the photograph is taken with the radioactive sample sandwiched against the emulsion of the film.
Thus, in autoradiography (in its narrow sense), the radioactivity of the material being tested is measured by a film sensitive to radioactivity.
In autofluorography, a fluor or scintillator, which is excited or stimulated by radioactivity to emit light, is applied in close proximity to the radioactive material and the intensity of light emission is measured by a photographic film, which is sensitive to light.
Autofluorography has important advantages over conventional autoradiography, the most important of which is a markedly shorter exposure time (typically shortened from two weeks to 16 to 24 hours) with weak radioactive emitters, such as tritium.
However, in spite of this important advantage, presently known autofluorographic techniques have serious disadvantages, particularly in systems where relatively thicker layers of absorbent or adsorbent materials are used in the separation process, e.g. polyacrylamide gel electrophoresis which is frequently used in receptor site, nucleic acid, and enzyme research.
One of the problems is developing a method for placing and maintaining the scintillator fluor in close proximity to the radioactive emitter. If not in close proximity, a portion of the emitted radioactive particles will not reach the scintillation fluor. In the case of thin layer chromatography, the scintillaor fluor can be dissolved in a suitable carrier, e.g. benzene or toluene, and then sprayed onto the thin layer separation medium, e.g. a paper strip, containing the radioactive sample. After drying, a piece of film sensitive to the light emission of the scintillator is then juxtaposed and this sandwich is allowed to stand for a time sufficient to achieve exposure. In such a system, it is difficult to evenly distribute the scintillator fluor, the radioactive material may spread and diffuse, and the small crystals of scintillator fluor tend to be so loosely bound that great care must be exercised in handling the sample.
In additon to the above disadvantages, it is sometimes desirable to use thicker layers of adsorbent or absorbent material. Once any appreciable thickness is used, i.e. greater than about 0.1 mm, the technique of spraying no longer places the scintillator fluor in close enough proximity to enough of the radioactive material with a drastic loss in the ability of the scintillator fluor to be excited by the emitted particles and convert them into light.
Accordingly, it is necessary to somehow transport the scintillator fluor into the interior of the separation medium. One method for accomplishing this transportation is by soaking the separation medium of absorbent or adsorbent material in a bath containing the scintillation fluor dissolved in a suitable carrier.
Two of the most common separation media used in electrophoresis are aqueous polyacrylamide and agarose gels. Gel electrophoresis is a method of separating charged particles, such as proteins, whereby the charged particles move through a gel medium under the influence of an applied electric field, their rate of movement through the lattice formed by the hydrated gel being dependent on charge and molecular size or weight. When the electric field is removed, the particles are present in the gel in discrete bands which can either be sliced up for liquid scintillation counting, or in the case of radionuclides such as tritium which emit lower energy particles, more preferably analyzed by autofluorography.
The technique now used most often to prepare polyacrylamide gels for autofluorography is described in Bonner and Laskey, Eur. J. Biochem., Vol. 46, pages 83-88, 1974, incorporated hereinby reference. In that method the radioactively-labelled protein is separated by electrophoresis using an aqueous polyacrylamide gel, followed by soaking the gel in about 20 times its volume of dimetylsulfoxide (DMSO) for 30 minutes, and then immersed a second time for 30 minutes in fresh DMSO to displace all the water from the gel. The next step is to soak the gel in a 20% (w/w) solution of 2,5-diphenyloxazole (PPO) in DMSO to impregnate the gel with scintillator which is then precipitated in the gel by washing with water. The gel is finally dried and exposed to the film. This technique has numerous disadvantages, many of which are reported in the appendix of an article by Laskey and Mills in the Eur. J. Biochem., at Vol. 56, pages 335-341, 1975, incorporated herein by reference. Agarose gels containing less than 2% polyacrylamide (plus 0.5% agarose) or agarose alone dissolve in DMSO unless methanol is substituted for the DMSO. Even this substitution is only effecive for gels having less than 2% polyacrylamide, since gels having higher concentrations of polyacrylamide shrink severly when contacted with methanol. Even at 30% methanol, shrinkage of higher polyacrylamide concentration gels may take place. Another disadvantage is that the failure to remove all the DMSO may result in adhesion of the film to the gel and artefactual blackening of the film. Another disadvantage is the ability of DMSO to penetrate through the skin of anyone handling it by itself or the gel which has been soaked in it, thereby carrying dissolved material with it through the skin as well as imparting a garlic smell to the person's breath. Another disadvantage is that the gels must be soaked in the DMSO-fluor solution for as much as 3 hours to obtain complete impregnation. A further disadvantage is that high concentrations of PPO, concentrations between 14% and 19% (w/w) being typical, must be used in the impregnation solution. Another disadvantage with DMSO as well as with other conventional carriers is that while PPO is efficient in converting absorbed radiation into photons of light, it is somewhat limited in its ability to absorb the energy emitted by the radioactive emitter. Another disadvantage is that the soakings in DMSO to dehydrate the gel are time-consuming.
One method for increasing the absorption ability of PPO when thin layer chromatography is being employed is described in Bonner and Stedman, Analytical Biochemistry, Vol. 89, pages 247-256, 1978, incorporated herein by reference. Three methods for detection of .sup.3 H and .sup.14 C in silica gel thin layer chromatograms are described in that article. The first method utilizes 2-methylnaphthalene (2MN) which is described as being a scintillation solvent for use in solid systems by analogy to scintillation fluids which many times contain a solvent in addition to the scintillator. As in liquid systems, the solvent molecules collect the energy from the emitted beta radiation and transfer it to PPO molecules, which then emit photons of light. A solvent is a compound which converts the kinetic energy radiated by the radioactive emitter to electronic excitation energy and transfers that energy to the fluro dissolved therein. The first method comprises dipping the dried thin layer plates in a solution of 2MN which has been liquified by heating and which contains 0.4% (w/v) of PPO, until they are soaked and then removing the plates from the solution. When the solution has solidified, the plate is placed against film and exposed. An alternative, if spraying is deemed to be more desirable, is to replace 10% of the 2MN with toluene to make the solution a liquid at room temperature. The second method involves dipping the plates in an ether solution containing between 7% and 30% (w/v) of PPO, drying the plates and then exposing as above, with better sensitivity being seen as the PPO concentration increases. The third method involves dipping the thin layer plates in melted PPO until soaked, removing and then heating until the excess PPO has drained off, and exposing to film as above. While useful in thin layer chromatography, numerous problems exist in attempting to use such systems with other media, e.g. One problem is that neither PPO nor 2MN is soluble nor miscible in water to any appreciable extent. Accordingly aqueous polyacrylamide or agarose gels are not impregnated with PPO nor 2MN while in the hydrated state, nor even if dried since the lattice structure collapses upon drying. Secondly, PPO and 2MN are very expensive even if it were possible to use them in such systems. The second method also is not useful with aqueous gels since ether and similar solvents such as alcohols cause drastic shrinkage of such gels. Furthermore, relatively high (7% to 30%) concentrations of expensive PPO in the ether are required for efficient fluorography.